Pepino mosaic virus
(PepMV), a member of Potexvirus
genus, is a significant pathogen in greenhouse tomato production worldwide. To
better understand the virus and the disease it causes, we initiated the
molecular characterization of an Arizona isolate (PepMV-AZ).
Cloning and sequencing analysis of selected genomic regions revealed the
presence in this isolate of two distinct strains that share a sequence identity
of only 85%. One strain was similar to several isolates found in Europe with
sequence identities of ~99%, another strain was similar to the US1isolate with a
sequence identity of 99%. Only one of 96 clones sequenced was found to be a
recombinant between the two strains, indicating a very low frequency of
recombination in PepMV. To clone high-fidelity
infectious cDNA, purified viral RNA used as a template
to synthesize full-length infectious cDNA. The first
strand cDNA synthesis was primed by an oligo(dT)40 primer with a BamHI
site at its 5 end. The second strand cDNA synthesis
was carried out with the PfusionTaq
DNA polymerase, primed with an oligonucleotide that contain a T7 promoter
sequence at the 5 end and 32 nucleotides identical to the 5 terminal
sequences of a specific PepMV strain. The full-length
infectious cDNA was cloned into the puc18 plasmid.
Infectious transcripts were synthesized readily in vitro by T7 RNA
polymerase using linearized plasmids as templates.

Background

PepMV is an emerging pathogen that
has rapidly become endemic in greenhouse tomatoes1,2.
Since the first report of the virus on tomatoes3, PepMV has spread across the globe. Symptoms of PepMV on tomatoes vary from yellow or chlorotic spots, leaf
beaching, to fruit discoloration (Fig. 1).

Phylogenetic analysis grouped the 26 PepMV
genomic sequences available in Genbank into five
distinct, well supported clades (Fig. 3), with the exception of the apparently
recombinant US2 genome4. The sequence identities are greater than
99% within each clade except Ch2 where sequence identities ranges from 97 to
99%. Each clade possibly represents a strain with two or more member isolates.
This classification breaks the original Ch2 clade1 into a new Ch2
clade and a new Polish clade (PL).

Two PepMV genomic fragments from
an Arizona PepMV isolate were cloned by RT-PCR with
two pairs of primers conserved among PepMV isolates:
A 650 bp fragment near the 5 terminus and an
internal 1 kb fragment over the p26 ORF (Fig. 2A, 2B). Sequencing of over 80
random clones revealed the presence of two distinct populations of sequences:
those of the EU strain and of the US1 strain (Fig. 4). Only one recombinant
sequence was found, indicating a low recombination frequency in PepMV.

Fig. 4. The Arizona
isolate contains two PepMV strains. Sequences
from 13 randomly selected RT-PCR clones of the 1 kb region containing the p26
ORF of PepMV were aligned and analyzed phylogenetically as described in Fig. 3. Cymbidium
mosaic virus (CymMV, EF125180) was included as an outgroup. Two populations of PepMV sequences areevident, one similar to the EU strain
and another to the US1 strain. Clone Pep4250-11 is a recombinant consisting of
about 850 nucleotides from the US1 strain and at the 5 end and about 100
nucleotides from the EU strain at the 3 end.

In order to engineer PepMV
infectious cDNA clones with high fidelity, virions
were purified from PepMV-infected Nicotianaclevelandii (Fig. 1E). viral
RNA was then isolated (Fig. 2C) and reverse-transcribed into cDNA using Superscript III and BamdT,
an oligo(dT) primer with a BamHI site. The cDNA:RNA hybrid migrated similarly as dsDNA
of the same size (Fig. 2D). After digestion with RNaseH,
the second strand cDNA was primed with
oligonucleotides containing the T7 promoter sequence and sequences specific to
the two PepMV strains found in the Arizona isolate,
and then extended with the high fidelity Phusion DNA
polymerase. Because of the large amount of starting viral RNA, the finished dsDNA of the expected size was easily visualized (Fig. 2D).
Full-length cDNA was then ligated into the puc18
plasmid for propagation. BamHI-linearized
plasmids directed the in vitro synthesis of full-length PepMV transcripts (Fig. 2E).

Conclusions

The Arizona PepMV isolate
contained a mixture of two PepMV strains with
infrequent recombination. A protocol was developed that allows one step high
fidelity cloning and engineering of infectious cDNA
from large RNA viruses.